Glycosyltransferases are enzymes that catalyze the transfer of glycosyl residues from a donor molecule to a particular acceptor (22
). Lactic acid bacteria produce a wide variety of a particular group of glycosyltransferases: glucosyltransferases (GTFs) and fructosyltransferases (FTFs), which synthesize glucose and fructose polymers, respectively, from sucrose without the need of cofactors (9
). An interesting feature of FTFs and GTFs is their ability to synthesize oligosaccharides of different polymerization degrees when efficient acceptor molecules like maltose or lactose are added to sucrose in the reaction medium (8
). This is known as the acceptor reaction, and the enzymes vary in their efficiency to perform this reaction.
GTFs have been the subject of intensive research, particularly those involved in industrial dextran production such as dextransucrase (DS) or alternansucrase produced by Leuconostoc
), whereas GTFs from Streptococcus
spp. are important due to their role in dental plaque formation (1
). Depending on its specificity, a GTF may transfer glucose, building polymers with the main chain joined by α1-6, α1-3, or α1-4 linkages (7
). However, no crystal structure is available for GTFs with the exception of amylosucrase (30
), a GTF producer of an amylose-type polysaccharide. GTFs present a large size, between 155 and 200 kDa, and are organized in three domains, starting in the N-terminal end by the signal peptide and a variable region with an unknown function, followed by the catalytic domain, where the residues implicated in catalysis have been located. Finally, a C-terminal domain is involved in binding to the synthesized glucan.
It has been proposed by sequence analysis and analogy with α-amylases that GTFs present a circular permutation of a (β/α)8
barrel in the catalytic domain (5
). This contrasts with FTFs, which on average have one third the molecular mass of GTFs and are not organized in domains. Moreover, Pons et al. (27
) predicted a β-propeller model for FTFs. Among FTFs, inulosucrase produces inulin, a fructan polymer with β2-1 linkages, whereas levansucrase catalyzes a similar reaction resulting in a β2-6-linked fructose polymer known as levan. In both cases, branching can occur in β2-6 and β2-1. In spite of the growing importance of inulin and fructooligosaccharides in the food industry, little is known about the biochemistry and molecular biology of these enzymes (4
). Up to now, more than 19 bacterial FTFs have been reported in GenBank, but no crystal structure is available. FTFs are also common in fungi and plants.
The inulosucrase of Leuconostoc citreum
is a cell-associated enzyme. It has been characterized both in its cell-associated insoluble form and after solubilization by urea treatment. Unexpectedly, this FTF has a molecular mass of around 165 kDa, the highest reported for FTFs. In its cell-associated form, it is highly specific for polymer synthesis, with low levels of fructose transferred to maltose and lactose when added to the reaction medium. The synthesized polymer has an inulin-like structure with β2-1 glycosidic linkages, as demonstrated by 13
C nuclear magnetic resonance (25
In the present study, we report the identification and functional characterization of islA
, a gene encoding for inulosucrase from Leuconostoc citreum
CW28. From the analysis of the nucleotide and the predicted amino acid sequence, it was concluded that this is a natural chimeric enzyme with three domains: the first and third with high identity with alternansucrase, a GTF from Leuconostoc mesenteroides
NRRL B-1355 able to produce alternan, a dextran-type polymer with alternating α1-3 and α1-6 linkages (2
). The second domain, which is the catalytic one, has similarity with levansucrase and inulosucrase of several microorganisms. No similarities have been reported between GTFs and FTFs in spite of the already mentioned analogies.